Application of FBXO9 protein in preparation of pancreatic cancer resisting medicine

Abstract

The invention relates to the field of biomedicine, in particular to application of FBXO9 protein in preparation of drugs for resisting pancreatic cancer. Experiments find that FBXO9 can inhibit the growth of pancreatic cancer cells and the occurrence of tumors for the first time, FBXO9 inhibits the occurrence of tumors by adjusting immune response, and low expression of FBXO9 indicates poor prognosis of pancreatic cancer patients, indicating that FBXO9 protein can be used as a marker to judge the prognosis of pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of FBXO9 protein in preparing drugs against pancreatic cancer. Background Art

[0002] Immune checkpoint blockade (ICB), especially immunotherapy targeting PD-1 / PD-L1, has opened up a new model for anti-tumor treatment. This therapy can specifically activate anti-tumor immunity and inhibit the immune escape function of cancer cells. PD-L1, as a membrane protein, is abnormally expressed in a variety of tumor cells such as melanoma, acute myeloid leukemia, and colorectal cancer. PD-L1 can inhibit the activation and immune response of CD8+T cells through PD-1, thereby promoting cancer cells to escape immune surveillance. Studies have shown that anti-PD-L1 immunotherapy can produce strong anti-tumor immunity and cause tumor regression. However, in the clinical application of various cancers including pancreatic cancer, ICB therapy is only effective for 1 / 3 of patients. Therefore, a thorough elucidation of the regulatory mechanism of PD-L1 provides a basis for the prognosis prediction of pancreatic cancer immunotherapy.

[0003] More and more studies have shown that protein posttranslational modifications (PTMs) play an important role in anti-PD-L1 anti-tumor effects, especially protein ubiquitination. Ubiquitination plays a key role in a variety of cellular processes, including regulating protein stability and interactions, which to a certain extent restricts cell viability and differentiation, as well as innate immunity and adaptive immunity. Studies have shown that posttranslational ubiquitination and deubiquitination processes play a key role in regulating PD-L1 protein levels and tumor immunosuppression. For example, TMUB1 reduces PD-L1 stability by inhibiting the polyubiquitination of PD-L1 at K281 in the endoplasmic reticulum, thereby promoting PD-L1 degradation and tumor immune evasion.

[0004] FBXO9, also known as FBX9, is a member of the F-box protein family and is an adaptor subunit of the SCFs (SKP1-cullin-F-box complex) E3 ligase. It is reported that FBXO9 can regulate the expression of multiple proteins, such as FBXW7, Neurog2, DPPA5, PPARγ, and PRMT4. In current studies, FBXO9 is often used as a potential biomarker or therapeutic target for sepsis. In order to further expand the scope of application of FBXO9 protein, further research on other applications is needed. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of FBXO9 protein in the preparation of anti-pancreatic cancer drugs.

[0006] In a first aspect, the present invention provides use of FBXO9 protein in the preparation of an anti-pancreatic cancer drug, wherein the NCBI accession number of the mRNA of the FBXO9 protein is NM_012347.

[0007] The present invention studies the expression, biological effects and tumor inhibition of FBXO9 protein and finds that the expression level of FBXO9 in pancreatic cancer tissue is significantly lower than that in normal tissue, FBXO9 overexpression inhibits cell viability, while FBXO9 silencing enhances cell viability, and FBXO9 is closely related to CD8+T cells in PAAD. From the above analysis results, it can be seen that low expression of FBXO9 is related to the poor prognosis of pancreatic cancer patients, and FBXO9 protein can inhibit the occurrence of pancreatic tumors and promote anti-tumor immune effects, indicating that FBXO9 protein can be used to prepare anti-pancreatic cancer drugs, providing a new application direction for FBXO9 protein.

[0008] In another preferred embodiment, the drug is a drug that inhibits the proliferation and migration of pancreatic cancer cells.

[0009] In another preferred embodiment, the drug contains the FBXO9 protein or its encoding gene.

[0010] In another preferred embodiment, the drug further comprises a pharmaceutically acceptable carrier.

[0011] The second aspect of the present invention provides a recombinant vector comprising the coding gene of the FBXO9 protein, which is obtained by inserting the coding gene between BamHI and XhoI of the pcDNA3.1-3xFlag-C plasmid.

[0012] The third aspect of the present invention provides the use of the recombinant vector in the preparation of a drug for treating pancreatic cancer.

[0013] The fourth aspect of the present invention provides the use of the detection reagent for the expression amount of FBXO9 protein in the preparation of a pancreatic cancer prognosis product.

[0014] In another preferred embodiment, the product is a kit.

[0015] In another preferred embodiment, the detection reagent includes an antibody that specifically binds to the FBXO9 protein.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention discovered through experiments for the first time that FBXO9 can inhibit the growth of pancreatic cancer cells and the occurrence of tumors. FBXO9 inhibits tumor occurrence by regulating immune responses, and low expression of FBXO9 can be used as a marker to judge the prognosis of pancreatic cancer.

[0018] The present invention uses bioinformatics analysis of the GSE183795 cohort in the TCGA database and the GEO database to find that the expression level of FBXO9 in pancreatic cancer tissue is significantly lower than that in normal tissue, and low expression of FBXO9 is associated with a decrease in overall survival, indicating that low expression of FBXO9 is associated with a poor prognosis in pancreatic cancer patients. Through Panc-1 and Patu-8988 cell experiments, it was found that overexpression of FBXO9 inhibits cell viability, while silencing of FBXO9 enhances cell viability, indicating that FBXO9 inhibits the occurrence of pancreatic tumors. Using the TIMER database to analyze FBXO9, it was found that FBXO9 is closely related to CD8 + This suggests that FBXO9 plays an immunomodulatory role in the tumor microenvironment of pancreatic cancer and can promote anti-tumor immune effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : The figures are related to the low expression of FBXO9 and the poor prognosis of pancreatic cancer patients in the embodiments of the present invention; wherein, A is the result of analyzing the expression of FBXO9 mRNA in pancreatic cancer and normal control tissues by TCGA and GTEx databases; B is the result of analyzing the expression of FBXO9 mRNA in pancreatic cancer and control tissues by GSE183795; C is the result of survival analysis of FBXO9 expression in 177 pancreatic cancer tissues by TCGA; D is the result of survival analysis of FBXO9 expression in 139 pancreatic cancer tissues by GSE183795; E is the result of IHC detection of FBXO9 expression in pancreatic cancer tissues and adjacent tissues; F is the result of analyzing the expression of FBXO9 in pancreatic cancer tissues of 112 pancreatic cancer patients and 82 adjacent tissues; G is the result of FBXO9 expression in pancreatic cancer tissues of 82 pancreatic cancer patients and paired adjacent tissues; H is the result of survival analysis of FBXO9 expression in 112 pancreatic cancer tissues.

[0020] Figure 2 The graphs show the changes in proliferation ability of Panc-1 and Patu-8988 transfected with FBXO9 overexpression plasmids detected by CCK8; A is the result of Panc-1, and B is the result of Patu-8988.

[0021] Figure 3The graphs show the changes in the proliferation ability of Panc-1 and Patu-8988 after transfection of FBXO9 sgRNA using CCK8; A is the result graph of Panc-1, and B is the result graph of Patu-8988.

[0022] Figure 4 The figure is a result diagram of the scratch experiment analyzing the change of migration ability of Patu-8988 cells after transfection of FBXO9 overexpression plasmid; wherein, A is the result diagram of migration ability of Patu-8988 cells, and B is the quantitative diagram of A.

[0023] Figure 5 The figure shows the result of scratch wound assay to analyze the changes in the migration ability of Patu-8988 cells after transfection with FBXO9 sgRNA; wherein, A is the result of the migration ability of Patu-8988 cells, and B is the quantitative graph of A.

[0024] Figure 6 The results of Transwell experiment to detect the changes in cell migration and invasion of PATU-8988 cells after transfection of FBXO9 overexpression plasmid, wherein A is the result of migration and invasion of Patu-8988 cells, and B is the quantitative graph of Figure A.

[0025] Figure 7 The figure shows the results of Transwell experiment to detect the changes in cell migration and invasion of PATU-8988 cells after transfection with FBXO9 sgRNA, where A is the result of migration and invasion of Patu-8988 cells, and B is the quantitative graph of Figure A.

[0026] Figure 8 These are the relevant result graphs for constructing a mouse subcutaneous transplant tumor model with overexpression of FBXO9; A is the result graph of the growth of subcutaneous transplant tumor tissue, B is the result graph of the weight of mouse subcutaneous transplant tumor tissue, and C is the result graph of the volume size of mouse subcutaneous transplant tumor tissue.

[0027] Fig. 9 This is a flow chart of the subcutaneous injection of EV and FBXO9-overexpressing Panc-02 cells into C57BL / 6 mice, respectively; EV stands for empty vector.

[0028] Fig.10 The following are the analysis results of tumor tissue of C57BL / 6 mice; A is the tumor tissue size diagram, B is the tumor tissue weight curve result diagram, and C is the tumor tissue growth curve result diagram.

[0029] Fig.11The infiltration results of CD8+T cells in tumors detected by flow cytometry; A is the infiltration results of CD8+T cells in tumors of the EV group, B is the infiltration results of CD8+T cells in tumors of the FBXO9 group, and C is the quantitative graph of the infiltration results.

[0030] Fig.12 The figures are the apoptosis results of Panc-02 cells after co-culture with spleen cells of healthy C57BL / 6 mice; A is the apoptosis results of Panc-02 cells in the EV group, B is the apoptosis results of Panc-02 cells in the FBXO9 group, and C is the quantitative graph of apoptosis. DETAILED DESCRIPTION

[0031] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] The following embodiment of the present invention discloses the use of FBXO9 protein in the preparation of an anti-pancreatic cancer drug. The NCBI accession number of the mRNA of the FBXO9 protein is NM_012347, and the sequence of the sgRNA targeting the FBXO9 gene is shown in SEQ ID NO.1:

[0033] SEQ ID NO. 1:GATGTACATCTTCCGATGGG.

[0034] In the following examples, all cell lines used for cell culture and transfection were from the Cell Bank of the Chinese Academy of Sciences.

[0035] The experimental methods include:

[0036] Construction of overexpression vector plasmid: The FBXO9 gene was inserted between BamHI and XhoI of the pcDNA3.1-3xFlag-C overexpression vector plasmid to obtain the FBXO9 overexpression plasmid.

[0037] Cell culture and transfection: Human pancreatic cancer cells Panc-1, PaTu-8988, and mouse pancreatic cancer cells Panc-02 were cultured in DMEM medium containing 10% fetal bovine serum, and the incubator was set to 37°C, 5% CO2. Lipofectamine 3000 was used to transfect the designated overexpression or sgRNA plasmids when the cells reached 80% density, and the transfection efficiency was determined by analyzing the protein and mRNA expression levels by immunoblotting and fluorescence quantitative PCR.

[0038] CCK8 experiment: Digest and centrifuge the cells in the logarithmic growth phase, collect them, resuspend them in culture medium, and count the cells. Then, inoculate 3,000 cells per well in a 96-well plate, culture for 24, 48, or 72 hours, add 10 μL of CCK8 reagent solution to each well, continue incubation for 4 hours, and use an ELISA reader to detect the absorbance of each well at 450 nm.

[0039] Scratch test: cells were inoculated into 6-well plates for culture. When the cell density reached 80% to 90%, a 20μl pipette tip was used to gently scratch a line in the center of the 6-well plate. Under the microscope, a blank scratch area was observed to be in the shape of a line. Floating and scratched cells were washed away with PBS, and culture medium was added for culture or other experiments were performed. The wound healing was observed at 0 and 18 hours of treatment and photographed under a microscope.

[0040] Transwell experiment: First, collect the cells needed for the experiment, wash with PBS, centrifuge, resuspend the cells in serum-free medium, and count the cells. Then add 800 μl of serum-containing medium to the bottom of the 24-well plate, and add 2×10 4 cells and cultured for 18 hours. When testing the cell invasion ability, 100 μl of pre-cooled and diluted Matrigel matrix gel must be added to the chamber in advance, and then the cell suspension is added. After the final 18 hours of culture, the chamber was removed, and the non-migrating cells remaining in the chamber were wiped off with a cotton swab. After fixation with 0.3% paraformaldehyde for 30 minutes, crystal violet staining was performed for 15 minutes, and the migration and invasion of the cells were observed under a microscope after drying.

[0041] Animal experiments: All animal experiments were approved by the Animal Ethics Committee of Bengbu Medical College. First, a subcutaneous tumor transplantation model was established in nude mice or C57BL / 6 mice. 5×10 6 Human pancreatic cancer cells Panc-1 or mouse pancreatic cancer cells Panc-02 that stably overexpress FBXO9 were used. After the mice formed tumors, the following operations were performed: the living conditions of the mice were observed, and the weight changes of the mice were weighed and recorded; the changes in the size of the tumors were measured every week; after the mice formed tumors for 4 to 6 weeks, the tumor masses were removed and weighed; and the tumors of C57BL / 6 mice were subjected to flow cytometry analysis.

[0042] Animal husbandry and killing methods: provide animals with standard animal feed and water, use isoflurane anesthesia, kill rats by decapitation or mice by cervical dislocation, and the animal carcasses are handled uniformly by the school.

[0043] Flow cytometry: To detect cell apoptosis, cells were first collected by centrifugation at 1000 rpm / min for 5 minutes, washed twice with PBS, and then resuspended in 200 μL PBS. PE-annexin V and 7-AAD antibodies were added, and after incubation for 15 minutes, apoptotic cell populations were analyzed by flow cytometry. + At the T cell level, the tumor tissue was first ground, Ficoll solution was added, and PBMC was collected by density gradient centrifugation. PBMC was resuspended with 100 μL PBS, and CD3, CD4, and CD8 antibodies were added. After incubation in the dark for 30 minutes, the cells were washed with PBS and resuspended. The flow cytometer parameters were set and the flow cytometer was tested.

[0044] Collection of clinical data and immunohistochemistry of patients: Clinical data of pancreatic cancer patients were collected from the Department of Hepatobiliary Surgery and Department of Tumor Surgery of Bengbu Medical College, and statistical analysis was performed. Pathological wax block tissues of patients were collected from the Department of Pathology of Bengbu Medical College and cut into 4 μm slices. IHC staining was completed in an automatic staining system. After dewaxing, hydration, antigen repair and blocking, the slices were incubated with primary and secondary antibodies for staining. After DAB color development, the slices were blocked, observed under a microscope, and photographed to collect images, and IHC scores were performed on each slice.

[0045] Bioinformatics analysis: The UCSC XENA database (https: / / xenabrowser.net / datapages / ) and GEO database (www.ncbi.nlm.nih.gov / geo / GSE183795) were used to download and extract pancreatic cancer clinical data, and the expression difference of FBXO9 between pancreatic cancer and normal tissues was analyzed. Kaplan-Meier survival analysis was performed; TIMER (http: / / timer.cistrome.org / ) was used to analyze the correlation between FBXO9 and immune effector cells.

[0046] Statistical analysis: GraphPad Prism 8 software was used for statistical analysis, and unpaired T test and paired T test were used for comparison between the two groups. Kaplan-Meier analysis and log-rank Mantel-Cox test were used for patient survival analysis, and P<0.05 indicated a significant difference.

[0047] Results Analysis

[0048] 1. Low expression of FBXO9 is closely associated with poor prognosis in patients with pancreatic cancer

[0049] First, bioinformatics analysis was performed using the TCGA database and the GSE183795 cohort in the GEO database. The results showed that the expression level of FBXO9 in pancreatic cancer tissues was significantly lower than that in normal tissues. Figure 1 As shown in A and B.

[0050] The relationship between FBXO9 and the clinical prognosis of pancreatic cancer patients was further analyzed. The results showed that low expression of FBXO9 was associated with decreased overall survival rate. Figure 1 As shown in C and D. Subsequently, pathological data of pancreatic cancer patients were collected, and immunohistochemical detection of FBXO9 protein levels in pancreatic cancer and adjacent tissues was performed. The results showed that the expression level of FBXO9 in tumor tissues was lower than that in adjacent tissues. Figure 1 As shown in E, F, and G.

[0051] An analysis of the overall survival of pancreatic cancer patients showed that patients with low FBXO9 levels had significantly lower overall survival rates. Figure 1 In addition, it was found that there was a consistent association between lower FBXO9 expression and TNM stage and other clinical indicators, and the results are shown in Table 1. These findings suggest that low FBXO9 expression is associated with a worse prognosis in patients with pancreatic cancer.

[0052] Table 1 Clinical indicators of FBXO9 in pancreatic cancer

[0053]

[0054]

[0055] Note: ρvalues ​​are x 2 The test value, n represents the number of cases, Low represents low expression, and High represents high expression.

[0056] 2. FBXO9 can inhibit cell growth and tumorigenesis

[0057] Knockout and overexpression experiments were performed in Panc-1 and Patu-8988 cells, and changes in cell proliferation were detected. The results showed that FBXO9 overexpression inhibited cell viability, while FBXO9 silencing enhanced cell viability. Figure 2 and Figure 3 shown.

[0058] The specific process of the knockout experiment is as follows: cells were seeded into six-well plates and transfected with FBXO9 sgRNA using Lipofectamine 3000 transfection reagent. After 48 hours, the cells were collected and the expression level of FBXO9 was verified by qPCR and Western blotting.

[0059] The specific process of the overexpression experiment is as follows: cells were inoculated into six-well plates and transfected with FBXO9 overexpression plasmid using Lipofectamine 3000 transfection reagent. After 48 hours, cells were collected and the expression level of FBXO9 was verified by qPCR and Western blotting.

[0060] In order to evaluate the migration and invasion ability of cells, the transfected pancreatic cancer cells were subjected to scratch assay and Transwell assay.

[0061] First, human pancreatic cancer cells PaTu-8988 were collected, washed with PBS, centrifuged, and resuspended in serum-free medium, and the cells were counted. Then, 800 μL of serum-containing medium was added to the bottom of the 24-well plate, and 2×10 4 cells and cultured for 18 hours. When testing the cell invasion ability, 100 μL of pre-cooled diluted Matrigel matrix gel must be added to the chamber in advance, and then the cell suspension is added. After the final 18 hours of culture, the chamber was removed, and the non-migrating cells remaining in the chamber were wiped off with a cotton swab. After fixation with 0.3% paraformaldehyde for 30 minutes, crystal violet staining for 15 minutes, and after drying, the migration and invasion of the cells were observed under a microscope. The results showed that overexpression of FBXO9 can significantly reduce the migration and invasion ability of cells. Figures 4 to 6 shown.

[0062] To study the antitumor effect of FBXO9 in vivo, a subcutaneous transplant tumor model was constructed, and all animal experiments were approved by the Animal Ethics Committee of Bengbu Medical College.

[0063] Panc-1 cells overexpressing FBXO9 were subcutaneously inoculated in nude mice. Through the subcutaneous transplantation tumor model, it was found that the growth and size of tumor tissue in the FBXO9 overexpression group were significantly lower than those in the EV control group. Figure 7 and Figure 8 As shown, from the above experimental results, it can be seen that FBXO9 inhibits the occurrence of pancreatic tumors.

[0064] 3. FBXO9 activates anti-tumor immune response

[0065] In order to evaluate whether FBXO9 inhibits tumorigenesis by regulating immune response, we first used the TIMER database (http: / / timer.cistrome.org / ) to analyze the correlation between FBXO9 and various immune effector cells. We found that FBXO9 was associated with CD8 + FBXO9 is closely related to T cells and may play an immunomodulatory role in the tumor microenvironment of pancreatic cancer.

[0066] To further explore the role of FBXO9 in antitumor immune response, mouse pancreatic cancer Panc-02 cells overexpressing FBXO9 were inoculated into C57BL / 6 mice with normal immune function. Fig. 9 As shown, compared with the EV control group, Fig.10 As shown in the figure, it can be seen that the growth of the tumor in the FBXO9 overexpression group was significantly inhibited, and its tumor weight was also lower than that in the control group. Flow cytometry further showed that the CD8+T cell infiltration in the tumor tissue of the FBXO9 overexpression group was significantly increased, as shown in the figure. Fig.11 In addition, after 3 days of co-culture of FBXO9-overexpressing Panc-02 cells or EV control group cells with spleen cells of healthy C57BL / 6 mice, it was observed that the apoptotic rate of FBXO9-overexpressing Panc-02 cells was significantly increased compared with that of the EV control group, as shown in Figure 2. Fig.12 As shown. It can be seen that the above research results indicate that FBXO9 can promote anti-tumor immune effects.

[0067] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Obviously, various changes and modifications may be made to the present invention by those skilled in the art without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such changes and modifications if they fall within the scope of the technical equivalents of the present invention.

Claims

1. The use of FBXO9 protein in the preparation of a drug against pancreatic cancer, characterized in that: The NCBI accession number of the mRNA of the FBXO9 protein is NM_012347.

2. The use of the FBXO9 protein according to claim 1 in the preparation of an anti-pancreatic cancer drug, characterized in that: The drug is a drug for inhibiting the proliferation and migration of pancreatic cancer cells.

3. The use of the FBXO9 protein according to claim 2 in the preparation of an anti-pancreatic cancer drug, characterized in that: The medicine contains the FBXO9 protein or its encoding gene.

4. The use of the FBXO9 protein according to claim 3 in the preparation of an anti-pancreatic cancer drug, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

5. A recombinant vector comprising the gene encoding the FBXO9 protein of claim 1, characterized in that: The coding gene was inserted between BamHI and XhoI of pcDNA3.1-3xFlag-C plasmid.

6. Use of the recombinant vector according to claim 5 in the preparation of a drug for treating pancreatic cancer.

7. Use of the FBXO9 protein expression detection reagent of claim 1 in the preparation of a pancreatic cancer prognosis product.

8. The use of the FBXO9 protein expression detection reagent according to claim 7 in the preparation of a pancreatic cancer prognosis product, characterized in that: The product is a test kit.

9. The use of the FBXO9 protein expression detection reagent according to claim 8 in the preparation of a pancreatic cancer prognosis product, characterized in that: The detection reagent includes an antibody that specifically binds to the FBXO9 protein.

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